Native chemical ligation (NCL) is a tool for the total synthesis and semi-synthesis of full-length proteins with site-specific post-translational modifications (see Dawson, P. E., et al. Science 1994, 266, 776). Application of NCL utilizes access to an N-terminal Cys-containing peptide fragment and a peptide C-terminal α-thioester. After an initial transthioesterification, whereby the Cys side-chain displaces the thiol from the C-terminal thioester fragment, a spontaneous S-to-N acyl shift leads to the thermodynamically stable amide bond (see FIG. 1A).
Peptide fragments bearing an N-terminal Cys can be obtained by SPPS using a 9-fluorenylmethoxycarbonyl (Fmoc-) α-amine protecting group strategy (see Flavell, R. R., et al. Acc. Chem. Res. 2009, 42, 107; Dhall, A., et al. ACS Chem. Biol. 2011, 6, 987; and Weller, C. E., et al. Biopolymers 2014, 101, 144) or by heterologous expression in Escherichia coli (see Erlanson, D. A., et al. Chem. Biol. 1996, 3, 981). In contrast, the direct synthesis of peptide α-thioesters by Fmoc-chemistry is limited by their inherent lability toward the organic bases employed for Fmoc-deprotection. Peptide α-thioesters may indeed be synthesized with a C-terminal thiol resin-linker using the tert-butyloxycarbonyl (Boc-) α-amine protecting group strategy (see Camarero, et al. J. Pept. Res. 1998, 51, 303 and Hackeng, T. M., et al. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10068). However, applications of Boc-chemistry can have several limitations, including incompatibility with specific phosphorylated (see Otvos, L., et al. J. Pept. Protein Res. 1989, 34, 129) and glycosylated amino acids (see Gamblin, D. P., et al. Chem. Rev. 2009, 109, 131) and the use of hydrogen fluoride (HF) gas for peptide cleavage from the solid-phase (see Muttenthaler, M., et al. Nat. Protoc. 2015, 10, 1067). Recently, trifluoromethanesulfonic acid (TFMSA) has been reported as an alternative to HF, but the broad utility and functional group compatibility of TFMSA is currently unknown (see Gates, Z. P., et al. Chem. Commun. 2016, 52, 13979).
Common strategies to generate peptide α-thioesters generally use Fmoc-chemistry in conjunction with multi-step manipulation after SPPS (see Zheng, J. S., et al. Acc. Chem. Res. 2013, 46, 2475 and Blanco-Canosa, J. B., et al. J. Am. Chem. Soc. 2015, 137, 7197), or modification of the solid-phase linker prior to peptide assembly (see Erlich, L. A., et al. Org. Biomol. Chem. 2010, 8, 2392 and Ollivier, N., et al. Org. Lett. 2010, 12, 5238), each with its inherent limitations and synthetic challenges (see Mong, S. K., et al. ChemBioChem 2014, 15, 721). Several thioesterification strategies utilize modified C-terminal amino acids (see Erlich, L. A., et al. Org. Biomol. Chem. 2010, 8, 2392) or strongly acidic conditions and elevated temperatures with Cys (see Kang, J., et al. Org. Biomol. Chem. 2009, 7, 4918) to favor intramolecular N-to-S acyl shift of the backbone amide bond, followed by transthioesterification with external thiols. Functionalized resins containing alkyl thiols that are suitably poised for nucleophilic attack at the C-terminal amide bond (see Taichi, M., et al. Org. Lett. 2013, 15, 2620), also known as crypto-thioesters (see Sato, K., et al. ChemBioChem 2011, 12, 1840), hold promise due to the minimal chemical manipulation required post-SPPS (see Tailhades, J., et al. J. Pept. Sci. 2015, 21, 139). The complex chemistry required to install crypto-thioesters, however, can limit their accessibility to a handful of laboratories. Therefore, efforts to expand the utility of NCL may benefit from facile and high-yielding Fmoc-based strategies to access peptide α-thioesters.